Major progress has been made in the understanding and translational advancement of cell-based therapeutics for chronic ischemic heart disease, a condition affecting approximately 5 million Americans. We have used the first funding period of this award to develop sophisticated porcine models of ischemic cardiomyopathy characterized by progressive ventricular remodeling. Using this model we have tested several strategies for cell-based therapeutics, gleaned major mechanistic insights, and developed sufficient preclinical data to be able to initiate clinical trials. Importantly, we showed that engraftment and differentiation of mesenchymal stem cells (MSCs) in the injured porcine heart was accompanied by 1) gap junction formation with host cells, 2) stimulation of endogenous cardiac stem cell (CSC) proliferation and differentiation and 3) sustained reverse remodeling and contractile restoration in myocardial areas of prior chronic ischemic dysfunction. In preliminary findings, we also showed that combining MSCs with CSCs leads to substantially greater efficacy at infarct size reduction. In this renewal application, we propose to identify the molecular underpinnings of this novel cell- based therapeutic strategy for ischemic cardiomyopathy using our established porcine model. Accordingly, we propose a series of three aims designed to test novel mechanistic hypotheses regarding MSC and CSC-based cardiac regeneration. We propose to 1) establish the mechanisms underlying the improved infarct scar reduction resulting from a combination of autologous CSCs and MSCs, 2) assess the impact of cell-cell communication by connexin 43 (Cx43)-mediated gap junctions on cardiac regeneration, and 3) assess the cell- cell interaction and efficacy of pharmacologically and/or genetically modified CSCs for myocardial repair in ischemic cardiomyopathy. These studies will continue our trajectory of work designed to obtain the most robust preclinical data possible to enlarge our knowledge base of the mechanisms underlying cell-based therapy for chronic ischemic heart disease. The proposed studies are timely, warranted, and could have a major health impact by addressing an unmet need in a large population of patients at risk for progressive heart failure, lifelong disability, sudden cardiac death, and recurrent hospitalizations. The approaches employed are well validated by our group and include in vitro and in vivo experimental models and efficient cell delivery systems and state of the art imaging strategies currently in use by our group. Our mechanistic research program has high translational value and is highly significant, as cellular therapy offers the potential to regenerate new myocardium and decrease progression of heart failure in patients with left ventricular dysfunction due to healed myocardial infarction. Together, these aims will advance our understanding of the mechanisms underlying cell- based therapy for chronic ischemic heart disease, with specific emphasis placed upon the impact of a combination of cell types and modulation of molecular mechanisms that can have additive or synergistic properties, thereby enhancing the efficacy and durability of cell therapy for ischemic cardiomyopathy.